Ketolide antibiotics have recently attracted significant interest because they have activity against a number ofmacrolide-resistant microorganisms, in particular staphylococci and enterococci. The overall objective of this research program is to use combinatorial biosynthesis and biochemical pathway engineering to produce novel functionalized ketolide antibiotics. A combination of two technologies belonging to Virginia Commonwealth University (VCU), and Midwest Molecular (a small business located in Minneapolis, Minnesota) will be used to achieve this goal. The VCU technology comprises a gene cassette capable of in vivo generation of a range of novel hydroxylated and desaturated cyclohexanecarboxylic acid starter units for polyketide biosynthesis. The Midwest Molecular technology is a set of versatile pikromycin polyketide biosynthetic enzymes for production of 14-membered ketolide antibiotics that can be engineered to accept these novel starter units. PROPOSED COMMERCIAL APPLICATIONS: Ketolides hold promise as a solution to the increase in erythromycin- resistant bacterial pathogens, including Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus and some enterococci. Numerous large pharmaceutical companies have semisynthetic ketolides which are now in phase II clinical trials. The proposed research will use a genetic-based precursor-directed biosynthetic technology in combination with polyketide synthase genetic engineering, to generate in an economically viable manner a range of novel ketolides not available from other technologies.